1. Field of the Invention
This invention relates generally to compositions and methods of enzyme replacement therapy (ERT). More specifically, the invention is directed to compositions and methods for treatment of enzyme deficient disease such as hypophosphatasia using a genetically modified polynucleotide to produce in an active secretory form of alkaline phosphatase.
2. Description of the Related Art
Alkaline phosphatase (ALP) is a ubiquitous plasma membrane-bound enzyme. Hypophosphatasia is an inherited metabolic disorder of defective bone mineralization caused by deficiency of a form of ALP know as tissue-nonspecific alkaline phosphatase (TNSALP). Clinical severity is remarkably variable, ranging from death in utero to merely premature loss of dentition in adult life [1, 2]. Despite the presence of TNSALP in bone, kidney, liver, and adrenal tissue in healthy individuals, clinical manifestations in patients with hypophosphatasia are limited to defective skeletal mineralization that manifests as rickets in infants and children and osteomalacia in adults [2]. In the most pernicious form of hypophosphatasia, the perinatal lethal variant, profound skeletal hypomineralization results in caput membranaceum with shortened and deformed limbs noted. Some affected neonates survive for several days or weeks. They often succumb to respiratory failure brought on by pulmonary hypoplasia and structural failure of the weakened skeleton from demineralization [3].
Osteoblasts modulate the composition of the bone matrix, where they deposit mineral in the form of hydroxyapatite. Specialized buds from the osteoblasts' plasma membrane are called matrix vesicles (MVs). The initiation of matrix calcification by osteoblasts and chondrocytes appears to be mediated by release of MVs, which serve as a sheltered environment for hydroxyapatite crystal formation [4-7]. MVs are alkaline phosphatase enriched, extracellular, membrane-invested bodies. Inside MVs the first crystals of hydroxyapatite bone mineral are generated. TNSALP hydrolyzes inorganic pyrophosphate (PPi) to monophosphate (inorganic phosphate; Pi), which is important for growth of the hydroxyapatite crystal [4, 5, 8-10]. Thus ALP functions as an inorganic pyrophosphatase (PPi-ase) [14, 15]. PPi itself impairs the growth of hydroxyapatite crystals as an inhibitor of mineralization [8, 11-13]. Insufficient TNSALP activity fails to hydrolyze PPi and the resulting build-up of unhydrolyzed PPi in the perivesicular matrix inhibits the proliferation of pre-formed hydroxyapatite crystals beyond the protective confines of MV membranes.
The level of plasma PPi increases in hypophosphatasia [16-18]. Even in the absence of TNSALP, the other phosphatases (AMPase and inorganic pyrophosphatase) can hydrolyze PPi, supplying Pi for incorporation into initial mineral within MVs [19] but still be insufficient to remove excess PPi at the perimeter of MVs. Thus, despite TNSALP deficiency, initial mineral could form within MVs, while its propagation into perivesicular matrix would be inhibited by a local build-up of PPi [20, 21]. These findings suggest PPi as a plausible candidate as an inhibitor of mineralization and as a primary factor that causes clinical manifestations of hypophosphatasia.
Enzyme replacement therapy (ERT) has proven effective in preventing or reversing lysosomal storage in patients and animal models with lysosomal storage diseases (LSDs) [22-28]. Tremendous progress in the development of ERT has been made in the last three decades. Cellular uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself corresponding to oligosaccharide receptors on the target cells. Examples include the binding of high-mannose oligosaccharides of the enzyme to the mannose receptor (MR) and binding of phosphorylated high-mannose oligosaccharides of the enzyme to the cation-independent mannose 6-phosphate receptor (M6PR). Thus, LSDs have been considered potentially amenable to therapy with exogenously supplied enzymes.
The cell-specific delivery system was also designed to enhance the clinical effectiveness of ERT. In the case of Gaucher disease, delivery of the enzyme to the affected cells was achieved by modifying the N-linked carbohydrate on the enzyme. This exposed core mannose residues [29, 30], enabling the enzyme to bind to the MR, which is highly abundant on cells of the reticuloendothelial system [31, 32]. These findings led to clinical management of Gaucher disease by ERT [22]. Over 3,500 patients have been treated with dramatic clinical results [33].
However, hypophosphatasia caused by a deficiency of TNSALP seems to be a difficult disorder treated by ERT because TNSALP is a membrane-bound enzyme and is believed to require attachment at the cell surface to be functional. In fact, the results of multiple intravenous infusions of plasma ALP or purified liver ALP in patients with hypophosphatasia have been disappointing [34-38]. Administration of exogenous pyridoxal HCl delayed the onset of epileptic attacks and increased the life span of TNSALP−/− mice. Although the oldest survivor was 22 days old, all the homozygotes, however, died near weaning time, irrespective of their treatment regime [39].
The inventors have genetically engineered a Chinese Hamster Ovarian (CHO) cell line to produce a C-terminus-anchorless TNSALP enzyme, in secreted form, [40] and showed clinical effectiveness of ERT on hypophosphatasia mice. These results indicate that the C-terminus-anchorless membrane enzyme possesses the characteristics necessary for use in ERT where the membrane-binding form is ineffective. Deletion of the C-terminus membrane anchor will be applicable to other membrane-binding proteins whose deficiency leads to other human disorders including but not limited to paroxysmal nocturnal haemoglobinuria (PNH).
Targeted therapies have the advantage of reducing adverse effects on non-target organs as well as reducing the minimum effective systemic dose. Recently, Kasugai et al [41] has demonstrated that a small peptide consisting of a stretch of acidic amino acids (L-Aspartic acid or L-Glutamic acid) was selectively delivered to and retained in bone after a systemic administration. Furthermore, a small molecule, an estrogen, conjugated with an acidic-oligopeptide, has been selectively targeted to bone, leading to dramatic improvement of the bone mineral density in ovariectomized mice with no or few adverse effects to liver and uterus [42]. However, whether such a bone-targeting system with an acidic oligopeptide could be applied to a large molecule such as an enzyme in a manner such that the enzyme is functional and efficiently produced remains unsolved.
The inventors have sought to address the issue of enzyme replacement therapy using membrane bound enzymes genetically modified to be synthesized in an active secretory form. In particular the inventors have applied this method to TNSALP as a treatment for hypophosphatasia. This method of releasing membrane bound enzymes in a functional form will offer new avenues for therapeutic strategies to combat disease of enzyme deficiency.